Endotoxin has been recognized over the last ninety years as a potent immunoactivator. W. B. Coley reported in Ann. Surg. 14 199 (1891) the use of "Bacterial Toxins" in cancer treatment. It was also reported in JAMA 54 250 (1919) that the cure rate in inoperable patients utilizing such a mixed microbial vaccine was 4-7 percent. Endotoxin extracts were subsequently studied in several animal models. Gratia and Linz in Comp. Rend. Soc. De Biol.108: 427 (1931) described hemorrhagic necrosis and concommitant tumor regression in a transplantable liposarcoma model in guinea pigs. Other rodent tumor models followed: sarcoma 180 in mice (Shwartzman, G. and Michailovsky, N., Proc. Soc. Exp. Biol. Med. 29: 737 (1932)), Ehrlich carcinomas in mice (Berendt, M. J. and North, R. J., Exp. Med., 151: 69 (1980)), and transplantable tumors in rats (Berendt, M. J. and North, R. J., private communication.).
Recent work on the observation of tumor necrosis in endotoxin-treated animals indicates that the endotoxic fraction itself may not be directly responsible for necrosis (Carswell, E. A., Old, L. J., Kassel, R. L., Green, S., Liore, N., and Williamson, B., Proc. Nat'l Acad. Sci. USA 72: 3666 (1975)). Instead, necrosis formation may be mediated by a factor termed tumor necrosis factor (TNF) which has been isolated from mice previously stimulated by macrophage activators such as BCG, C. parvum or zymosan. In addition, TNF has been shown to be cytotoxic against certain tumor lines in vitro, and antitumor activity has been ascribed to this substance in vivo.
Prior to the present invention and during a search for microbial components having antitumor activity, it was found that when certain preparations of endotoxin were combined with trehalose dimycolate (TDM) and oil droplets and injected into established malignant line-10 tumors in Strain 2 guinea pigs, a high rate of cures and systemic tumor immunity developed. This led to a reinvestigation of the value of endotoxin as an immunotherapeutic agent. The most powerful endotoxin adjuvants were phenol-water (PQ) or chloroform methanol (CM) extracts from Re (heptoseless) mutant, gram-negative bacteria. These extracts contained endotoxic lipopolysaccharides (LPS) which made up phenol-water extracts from wild-type bacteria. Both ReG1 and lipipolysaccharide when injected in combination with TDM and oil droplets caused a rapid developing Shwartzman-like necrotic reaction in the tumors. Following this reaction, the LPS combination led to only a partial regression of injected tumors, and their growth continued after about two weeks. In contrast, injection of the ReG1-TDM combination led to high rates of permanent regression and development of systemic immunity against a challenge with line-10 tumors. Tumor regression with ReG1+TDM or CWS+TDM and more advanced tumors could be treated with greater success.
In a paper by Dr. J. L. Cantrell et al appearing in Cancer Research 39, 1159-1167, April (1979) it was disclosed that a combination of chemotherapy and immunotherapy are highly effective in causing regression of an established tumor in mice, whereas either treatment alone was ineffective. In this study, the immunotherapy used involved injection of KC1 - extracted tumor antigens in oil-in-water emulsions with or without trehalose dimycolate.
Also in U.S. Pat. Nos. 4,436,727 and 4,505,903 various combinations of refined detoxified endotoxin or purified pyridine soluble extracts of microorganisms with cell wall skeleton and/or trehalose dimycolate were disclosed as being useful in the treatment of cancerous tumors. However, prior to the present invention, immunotherapy was performed with biological response modifiers, as non-specific immunotherapy or with tumor antigens alone. However, non-specific immunotherapy had only a short effect on tumors and tumor antigens were low in immunogenicity. In addition, the adjuvants previously available for use in human vaccine had low activity, and hence the immunotherapy was not entirely satisfactory. Thus, while there is considerable prior art on adjuvants and on tumor antigens, there is no prior art on the use of non-toxic biological adjuvants to enhance protective immunity when used in combination with tumor antigens. Therefore, what was needed was a potent but non-toxic immunostimulant which could be utilized in conjunction with tumor associated antigens to provide a protective and lasting tumor immunity.
Accordingly, one or more of the following objects will be achieved by the practice of this invention. It is an object of this invention to provide a vaccine which is effective in thie treatment and prevention of tumors. Another object of this invention is to provide a vaccine comprised of non-toxic and highly effective adjuvants from microbial sources and tumor antigens. A further object of this invention is to provide a method for enhancing the antitumor activity of immunogenic tumor antigens. Another object of this invention is to provide a process for the preparation of tumor vaccines. A still further object is to provide a process for the preparation of tumor vaccines comprised of adjuvants and tumor antigens. Another object is to provide a process for the preparation of vaccines comprised of refined, detoxified endotoxins and tumor antigens. A further object is to provide a method for using the vaccines in the treatment and prevention of tumors. These and other objects will be readily apparent to those skilled in the art in light of the teachings contained herein.